Peptide having ability to activate cancer-related gene

ABSTRACT

To provide a cancer diagnostic reagent for determining malignancy of a cancer patient or a cancer cell and a tendency of canceration of a healthy subject, the reagent including a peptide having an ability to activate a cancer-related gene and extracted from cell membrane surfaces of human squamous-cell carcinoma cells or including a synthetic polynucleotide encoding the peptide or a partial amino acid sequence of the peptide.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to peptides activating a cancer-relatedgene and derived from cell membrane surfaces of human cancer cells,immunosuppressive agents including the peptides as an effectivecomponent, and anticancer agents including antibodies against thepeptides as an effective component. The present invention furtherrelates to diagnostic reagents including polynucleotides encoding thepeptides for diagnosing malignancy of cancer or tendency of canceration.Furthermore, the present invention relates to methods for manufacturingthe peptides.

2. Description of the Related Art

Cancer is a disease caused by cells which started unregulated growth.The cells continue to proliferate and infiltrate into neighboring normalcells to destroy the normal functions. Some of the cells spread bymetastasis from their original site to one or more sites elsewhere inthe body, leading to a loss of normal cell functions and to depressionof functions of organs. Thus, cancer is a disease that leads patients todeath. Normal cells proliferate but will stop growing when they come incontact with solid substances (contact inhibition). On the other hand,the contact inhibition property is lost in cancerous cells.Consequently, the cancerous cells continue to grow in their host as longas the host is alive. Among cells extracted from the body, cells havingability for continuing unlimited growth can be established as a cellline. Cancer, in a broad sense, is a state of uncontrolled cell growthdue to mutation of a gene, in particular, due to mutation of a genepositively or negatively regulating cell growth. Heretofore, manystudies have been conducted on genes contributing to carcinogenesis or agrowth mechanism and peptides or proteins relating to them (JapaneseUnexamined Patent Application Publication Nos. 2003-517306 and2000-217585).

Among peptides or proteins derived from cell membrane surfaces of humancancer cells, generally, HLA-binding peptides are particularly thoughtto be cancer antigens and are thought to act on immunocompetent cells asantigens, most of all, as immunogens. Additionally, it is widely knownthat some proteins and glycoproteins derived from cancer cells andimmunocompetent cells such as macrophages isolated from a cancer-bearingliving body have an immunosuppressing activity preventing thedestruction of cancer cells, for example, immunosuppressive acidicprotein (IAP). Such proteins and glycoproteins are clinically used formeasuring the degree of immunosuppression.

Heretofore, it has been thought that cancer cell growth progresses by amechanism due only to self division of cancerous cells. However, therate of cancer cell growth is not constant and cancer cells rapidlyproliferate at some point. Such phenomena cannot be fully explained bythe conventional view only.

SUMMARY OF THE INVENTION

On the basis of the above-mentioned phenomenon that cancer rapidlyprogresses at some point, cancer cells have a possibility of secreting afactor outside the cancer cells. Such a factor further accelerates thecancer cell growth by acting on normal cells neighboring the cancercells so as to activate a cancer-related gene in the normal cells. Ifthis factor is found, not only a mechanism of progression of cancer isclarified but also a useful drug can be provided.

Namely, it is an object of the present invention to provide a factorhaving a function of accelerating cancer cell growth by acting on normalcells neighboring the cancer cells so that a cancer-related gene in thenormal cells is activated and to provide a corresponding gene of thefactor. It is another object of the present invention to develop avaluable use of the factor and its corresponding gene by verifying thefactor for the cancer-related-gene-activating function in normal cells.

The present inventor has performed intensive studies for overcoming theabove-mentioned problems, and, as a result, found a peptide and a geneencoding the peptide in an extract of a human squamous-cell carcinomacell line UTC-8 which has a high activity of metastasis. Furthermore,the inventor has proved that the peptide has a function of activating acancer-related gene in normal cells neighboring cancer cells by actingon the normal cells and accelerating cancer growth, and has proved thatthe peptide can be used as an immunosuppressive agent. In addition, theinventor has found that a polynucleotide capable of hybridizing with thegene encoding the peptide can be used as a reagent for diagnosingmalignancy of cancer or measuring tendency of canceration (easiness ofconversion to cancer: a degree of risk of canceration), which was notincluded in clinical examination items before, and that an antibodyagainst the peptide has an anticancer activity. Thus, the presentinvention has been accomplished.

The present invention relates to aspects (1) to (10) described below:

(1) A peptide having an ability to activate a cancer-related gene,wherein the peptide is derived from cell membrane surfaces of humansquamous-cell carcinoma cells and includes an amino acid sequencerepresented by SEQ ID No: 1 or an amino acid sequence having deletion,substitution, or addition of one or several amino acids in the aminoacid sequence represented by SEQ ID NO: 1;

(2) A peptide having an ability to activate a cancer-related gene,wherein the peptide is derived from cell line UTC-8 (FERM BP-08611)established from human squamous-cell carcinoma; and the peptide shows adetectable peak at a detection wavelength of 214 nm in gel filtration ofan extract from the cell line;

(3) An immunosuppressive agent comprising the peptide according to theaspect (1) or (2) as an effective component;

(4) An antibody against the peptide according to the aspect (1) or (2);

(5) A diagnostic reagent including the antibody according to the aspect(4) for determining tendency of canceration or malignancy of cancer;

(6) An anticancer agent comprising the antibody according to the aspect(4) as an effective component;

(7) A polynucleotide including a nucleotide sequence encoding at leastthree contiguous amino acids of the amino acid sequence of the peptideaccording to the aspect (1);

(8) A diagnostic kit for determining tendency of canceration ormalignancy of cancer, wherein the kit includes a peptide according tothe aspect (1) or (2) and a polynucleotide according to the aspect (7);

(9) The diagnostic kit according to the aspect (8) further including atleast one marker gene selected from the group consisting of:

Ras oncogene family;

v-crk avian sarcoma virus CT10 oncogene homolog-like lactatedehydrogenase B;

Placental growth factor;

Interleukin 8;

MAS1, activator of S phase kinase;

v-raf;

v-fms;

v-rel;

v-src;

GRO1;

Hepatoma-derived growth factor;

Vascular endothelial growth factor;

Bone morphogenic protein 3;

Squamous-cell carcinoma antigen recognized by T cell;

Interleukin-1 beta;

Conserved gene amplified in osteosarcoma; and

Lymphoid blast crisis oncogene; and

(10) A method for preparing a peptide according to the aspect (1) or(2), the method including the steps of immersing cells obtained byculturing human squamous-cell carcinoma cells in a culture medium into acitrate-phosphate buffer having a pH of 3.3 to 3.4 for extraction; andfractionating the extract by gel filtration using a citrate-phosphatebuffer having a pH of 6.8 to 7.0 for yielding a peptide having anability to activate a cancer-related gene in human normal cells.

The peptide according to the present invention is derived from cellmembrane surfaces of human squamous-cell carcinoma cells. In particular,the peptide is extremely remarkable in the fact that it has ability toactivate a cancer-related gene in human normal cells. Since thesensitivity of cells and tissues against this peptide of the presentinvention can be an indicator to predict canceration tendency of normalcells or progress of cancer, the peptide can be a useful diagnosticreagent for determining tendency of canceration or malignancy of cancer.Additionally, it is strongly suggested that the unregulated growth ofcancer cells is caused by, not only cancer cells themselves, but alsothe interaction accompanying the activation of various cancer-relatedgenes in normal cells. Thus, an entirely novel mechanism of cancergrowth is presented.

Therefore, the present invention can contribute greatly to the researchand development of diagnosis and therapy of cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are elution patterns of gel filtration of peptidesextracted in Example 1 according to the present invention. FIG. 1A showselution patterns of the peptides by using a citrate-phosphate bufferhaving a pH of 7.2 as an elution buffer and detection wavelengths of 280nm and 210 nm. FIG. 1B shows elution patterns of the fraction (elutionvolume: 21 ml) of the rightmost peak of the elution pattern using thedetection wavelength of 280 nm in FIG. 1A by using a citrate-phosphatebuffer having a pH of 6.8 as an elution buffer and detection wavelengthsof 280 nm and 214 nm.

FIGS. 2A and 2B are electrophoresis photographs showing gene patternchanges in cells treated with a peptide of the present invention whichwere investigated by conducting a PCR reaction using cDNA prepared byreverse transcription of total mRNA derived from the cells treated withthe peptide as a template and using DNA encoding the full length of thepeptide of the present invention as a primer.

FIGS. 3A and 3B are electrophoresis photographs showing gene patternchanges in cells treated with a peptide of the present invention. Thegene was prepared by a PCR reaction using cDNA prepared by reversetranscription of total mRNA derived from the cells treated with thepeptide as a template and using DNA encoding a sequence consisting ofthe first to seventh amino acids of the peptide of the present inventionas a primer.

FIG. 4 is a photograph showing the result when the monoclonal antibodyagainst the peptide of the present invention was subjected to acytotoxic test using cancer cells.

FIG. 5 is a diagram showing an immunosuppressive activity of the peptideof the present invention in a transplantation test.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The peptide according to the present invention has a function ofactivating a human cancer-related gene and includes the following aminoacid sequence:Gln-Pro-Gln-Phe-Gly-Arg-Arg-Met-Glu-Ser-Lys (SEQ ID NO: 1)

Additionally, a peptide including an amino acid sequence havingdeletion, substitution, or addition of one or several amino acids in theamino acid sequence represented by SEQ ID NO: 1 is included in thepresent invention as long as the peptide has the function of activatinga cancer-related gene.

The above-mentioned cancer-related gene refers to not only oncogenes butalso immune system genes. The activation of a cancer-related gene meansacceleration of canceration or cancer growth through activation of anoncogene or through inactivation of a tumor suppressor gene and/oractivation or inactivation of an immune system gene, e.g., a cellularimmunity system and/or humoral immunity system.

The immunosuppressive activity of the peptide according to the presentinvention is notable. For example, it is recognized that the peptidesuppresses the rejection in tissue transplantation. Therefore, thepeptide of the present invention can be used as an immunosuppressiveagent.

The peptide represented by SEQ ID NO: 1 is derived from cell membranesurfaces of human squamous-cell carcinoma cells and can be extractedfrom human squamous-cell carcinoma cell line UTC-8 (Deposition No.: FERMBP-08611, which is deposited with International Patent OrganismDepositary, National Institute of Advanced Industrial Science andTechnology) by using a citrate-phosphate buffer having a pH of 3.3 to3.4.

More specifically, after the extraction by the citrate-phosphate bufferhaving a pH of 3.3 to 3.4, the extract is further applied to gelfiltration using a citrate-phosphate buffer having a pH of 6.8 to 7.2 asan elution buffer at a column flow rate of 0.1 to 0.28 ml/min, and thena fraction having a peak in a detection wavelength of 214 nm is isolatedto yield the peptide represented by SEQ ID NO: 1.

The peptide represented by SEQ ID NO: 1 of the present invention ischaracterized by the fact that the peak can be separated and detectedonly when a detection wavelength of 214 nm is used. The peak of thepeptide of the present invention cannot be detected when a detectionwavelength of 254 to 257 nm or of 280 nm, which are generally used inpeptide detection, is used.

In addition to the above-mentioned extraction method, the peptideaccording to the present invention can be prepared by conventionalchemical synthesis of peptides on the basis of the amino acid sequence.Namely, an amino acid derivative having a carboxyl group and aside-chain functional group being protected by a protecting group and anamino acid derivative having an amino group and a side-chain functionalgroup being protected by a protecting group are condensed in thepresence of carbodiimide or the like. Then, the protecting group for theamino group is removed to bind a next amino acid derivative protected bya protecting group. Such a reaction can be performed by a liquid-phasemethod or a solid-phase method. In general, the solid-phase method isused, except when a relatively large amount of peptide is synthesized.Particularly, in a method developed for identifying an antigenicdeterminant defined by its amino acid sequence, a spacer arm having anamino group on its tip is used. The spacer arm is prepared by chemicallytreating a cellulose film or the tip of a plastic pin. In the lattermethod, a peptide chain is extended by sequential reactions of solutionsin a 96-well plate; this method is called multi-pin peptide synthesis.

Furthermore, in another method, a peptide of the present invention maybe prepared by chemically synthesizing a DNA encoding the peptide byusing a DNA synthesizer; preparing a recombinant vector by connectingthe DNA to an appropriate expression vector; introducing the vector intoa host such as Escherichia coli; culturing the host; and collecting thepeptide of the present invention from the culture.

The peptide of the present invention is useful by itself and can be usedas a diagnostic reagent for determining tendency of canceration(easiness of conversion to cancer: a degree of risk of canceration) ofnormal cells or tissues, for determining whether or not a subject issuffering from cancer, and for determining malignancy of cancer.

The above-mentioned various determinations are performed by bringing thepeptide of the present invention in contact with a human cell or tissuespecimen obtained by surgical resection or biopsy, and utilizing thiscontact for detecting a cancer-related gene or for determining a changein the expression degree or the expression pattern of the gene. Thesensitivity of cells or tissues against the peptide of the presentinvention reflects, for example, a tendency of canceration (easiness ofconversion to cancer: a degree of risk of canceration) of the cells ortissues or a degree of malignancy of cancer.

In the determination of the degree of malignancy of cancer cells ortissues, the amount of expression of the gene encoding the peptide ofthe present invention may be directly determined without the contact ofthe peptide of the present invention with the cells or tissues. However,it is desirable that the cancer cells or tissues are brought intocontact with the peptide of the present invention, in order to achievehigher sensitivity in measurement.

Additionally, as is clear in diagnosis methods 1 and 2 described below,a polynucleotide (DNA and RNA) encoding at least three contiguous aminoacids of the amino acid sequence of the peptide of the present inventioncan be used as a diagnostic reagent for determining tendency ofcanceration (easiness of conversion to cancer: a degree of risk ofcanceration) of normal cells or tissues, for determining whether or nota subject is suffering from cancer, and for determining malignancy ofcancer. A diagnostic kit of the present invention preferably utilizessuch a polynucleotide in combination with a peptide of the presentinvention.

Diagnosis methods using the peptide of the present invention will now bedescribed.

[Diagnosis Method 1]

First, a specimen such as cells and tissues obtained from a subject tobe tested is brought into contact with the peptide of the presentinvention, and they are cultured for a predetermined period of time.Examples of the specimen include cells and tissues obtained from a highrisk group for cancer, such as subjects living in area polluted with achemical or radioactive material and smokers; cells and tissues whichare suspected to be a precancerous stage, such as polyp; cells andtissues neighboring cancer cells; cells and tissues at a region under adanger of metastasis of cancer; and peripheral blood.

When the specimen has a tendency of canceration, even if the cells andtissues are normal now, a change in an expression pattern of the gene isobserved: the gene encoding the peptide of the present invention isdetected, or the amount of expression of the gene is increased. When themalignancy of cancer is high, the amount of expression of the gene isfurther increased. Therefore, the tendency of canceration or malignancyof cancer can be determined by detecting the gene or determining thedegree of its expression.

In this diagnosis method, the cells or tissues of interest are broughtinto contact with the peptide, and then total mRNA is extracted from thecells or tissues. After synthesis of cDNA by reverse transcription usingthe mRNA as a template, PCR is further conducted by using the resultingcDNA as a template. In the latter PCR, all polynucleotides encodingsequences consisting of at least three contiguous amino acid residues ofthe amino acid sequence represented by SEQ ID NO: 1 are collectivelyused as primers. The polynucleotide may encode the full-lengthpolypeptide.

This diagnosis method will now be specifically described.

First, total mRNA is extracted by a common method from a specimen whichis prepared by treating normal tissues or cells with the peptide. Then,cDNA is synthesized by reverse transcription using the total mRNA astemplates to generate a cDNA pool which will be used as templates.

DNA encoding a sequence consisting of, for example, the first to fourthamino acids in the amino acid sequence represented by SEQ ID NO: 1 hasthe following nucleotide sequence:

CAR CCN CAR TTY (SEQ ID NO:2)

(wherein N is A, T, C, or G, R is G or A, and Y is C or T). Each of thenucleotide sequences, i.e., 32 types of DNA, is synthesized, and a poolincluding each DNA in an equal amount is used as forward primers.

Since the cDNA used as the template has a poly-T sequence correspondingto poly-A tailing of mRNA, an oligo-dT primer (poly-A primer) is used asa reverse primer.

The PCR using the cDNA pool as the templates is conducted by using theabove-mentioned forward primers and the reverse primer, followed byelectrophoresis and fluorescence staining.

When a change in an expression pattern of genes including the geneencoding the peptide of the present invention is induced by treating aspecimen with the peptide of the present invention, it is indicatedthat-the specimen is sensitive to the treatment with the peptide of thepresent invention; thus, it is determined that the specimen has a hightendency of canceration even if the cells or tissues as the specimen arecurrently normal. When a specimen is already cancerous, a change in thegene expression pattern increases with a degree of the malignancy.Therefore, the degree of the malignancy can be determined by measuringthe change.

The amount of the peptide expression can be determined by measuring thefluorescence intensity of the band in the electrophoresis. In order toprecisely determine the amount of the peptide expression, for example,quantitative PCR may be performed.

The quantitative PCR can be performed by, for example, real-time PCRusing the cDNA pool as templates, the above-mentioned primers, and afluorescent dye or a fluorescence-labeled probe which binds withdouble-strand DNA. Then, a relationship between the number of the PCRcycles and the fluorescence intensity is determined. By comparing theresults with those obtained by using a standard, the amount of the cDNA,i.e., the expression amount of the peptide of the present invention, canbe quantitatively determined.

A change in the gene expression pattern or an increase in the peptideexpression amount can be determined by comparing the expression level ofa control. As the control, the results obtained by conducting the sameprocedure as above using the same cells except that the peptide of thepresent invention is not used and/or the results obtained by treatingnormal cells or tissues that are completely free from cancer risk withthe peptide and conducting the same procedure as above, are used.

The normal cells and tissues that are free from cancer risk are derivedfrom young subjects. As such cells, for example, a kidney mesangium cell(ACBRI-1376; Applied Cell Biology Research Institute), a skin fibroblastcell (Catlog2F0-C25; Cell Systems), and a pancreatic epithelial cell(CBRI515; Applied Cell Biology Research Institute) are commerciallyavailable.

From the views described above, it is obvious that a combination of thepeptide of the present invention and the group of the above-mentionedprimers, i.e., the group of polynucleotides encoding sequencesconsisting of at least three contiguous amino acid residues of the aminoacid sequence represented by SEQ ID NO: 1, is useful as a cancerdiagnostic reagent kit for determining a tendency of canceration ormalignancy of cancer.

[Diagnosis method 2]

In another diagnosis method using the peptide of the present invention,cancer-related gene is used as a marker gene.

In this diagnosis method, cells or tissues of interest are treated withthe peptide of the present invention as in the above-mentioned diagnosismethod 1; thus the same specimen as in the diagnosis method 1 can beused. However, in this diagnosis method, the reverse transcription isconducted to generate a cDNA (first-strand cDNA) pool, which iscomplementary to the mRNA, by using total mRNA extracted from the cellsor tissues treated with the peptide as a template and using oligo-dTprimer having T7 RNA polymerase promoter region as a reverse primer.Then, after the second-strand cDNA synthesis, in vitro transcription(IVT) was conducted using T7 RNA polymerase and biotinylated rNTPs at37° C. for 14 hr to yield biotin-labeled cRNA. The amount of the cRNA ismeasured with a spectrophotometer to confirm that at least 10 μL of cRNAcan be yielded.

The diagnosis method further includes processes for bringing this cRNAlabeled with fluorescence dye or the like into contact with cDNA of acancer-related gene which is immobilized on a plate for hybridizationand for measuring the fluorescence intensity.

When the cRNA is hybridized to some cancer-related gene, it is suggestedthat the cancer-related gene is being expressed in the cells or tissues.When the fluorescence intensity of some cancer-related gene is increasedor decreased, it is suggested that the expression amount of thecancer-related gene is increased or decreased by the treatment with thepeptide of the present invention. Namely, for example, when theexpression of an oncogene is increased or the expression of an immunesystem gene which suppresses canceration is decreased, it is suggestedthat the tendency of canceration is high. Thus, on the basis of theexpression condition of a cancer-related gene, a tendency of cancerationor malignancy of cancer can be determined.

An increase or decrease in the expression amount of a cancer-relatedgene is determined by comparing the expression level in a control. Thecells and tissues used as the control are the same as those used in thediagnosis method 1.

As described above, immune system genes relating to cancer, in additionto oncogenes, are included in the cancer-related genes in thisspecification. Examples of such genes are as follows:

Ras oncogene family;

v-crk avian sarcoma virus CT10 oncogene homolog-like lactatedehydrogenase B;

Placental growth factor;

Interleukin 8;

MAS1, activator of S phase kinase;

v-raf;

v-fms;

v-rel;

v-src;

GRO1;

Hepatoma-derived growth factor;

Vascular endothelial growth factor;

Bone morphogenic protein 3;

Squamous-cell carcinoma antigen recognized by T cell;

Interleukin-1 beta;

Conserved gene amplified in osteosarcoma; and

Lymphoid blast crisis oncogene.

At least one gene of these cancer-related genes is used in thisinvention. For higher precision, yet more cancer-related genes may beused. In such a case, it is preferable to use a combination of genes inthe following groups as a genes set for diagnosis.

(a) Oncogene expression-increasing group: a gene encoding the peptide ofthe present invention and Ras oncogene and c-fos oncogene, etc.;

(b) Immune gene expression-increasing group: Interleukin-1 beta, etc.;and

(c) Immune gene expression-decreasing group: MHC class II, DM, andalpha, beta protein gene and killer cell lectin-like receptor subfamilyB, M member 1 gene.

Tables 1 and 2 show examples of cancer-related genes of which expressionis increased and immune system genes of which expression is increased ordecreased by the treatment with the peptide (HPLC-purified peptide)derived from cell membrane surfaces of human squamous-cell carcinomacells UTC-8 (Deposition No.: FERM BP-08611, which is deposited withInternational Patent Organism Depositary, National Institute of AdvancedIndustrial Science and Technology). TABLE 1 Cancer-related gene enhancedby the HPLC-purified peptide Gene Name Fold Change Common DescriptionProduct K03218 3.912448639 SRC; ASV; SRC1; v-src sarcoma (Schmidt-RuppinA-2) proto-oncogene tyrosine-protein c-SRC; p60-Src viral oncogenehomolog (avian) kinase SRC NM_001201 3.73617959 BMP3 bone morphogeneticprotein 3 bone morphogenetic protein 3 (osteogenic) (osteogenic)precursor D16431 3.595973492 HDGF hepatoma-derived growth factorhepatoma-derived growth factor (high-mobility group protein 1-like)(high-mobility group protein 1-like) X54936 3.576227427 PGF placentalgrowth factor, vascular placental growth factor, vascular endothelialgrowth factor-related endothelial growth factor-related protein proteinM95712 3.033826351 BRAF v-raf murine sarcoma viral oncogene v-raf murinesarcoma viral oncogene homolog B1 homolog B1 NM_005850 3.023255825 SF3B4splicing factor 3b, subunit 4, 49 splicing factor 3b, subunit 4 kDaNM_005937 2.995664358 MLLT6 myeloid/lymphoid or mixed-lineagemyeloid/lymphoid or mixed-lineage leukemia (trithorax homolog, leukemia(trithorax homolog, Drosophila); translocated to, 6 Drosophila);translocated to, 6 NM_001419 2.950166225 ELAVL1 ELAV (embryonic lethal,abnormal ELAV-like 1 vision, Drosophila)-like 1 (Hu antigen R) NM_0026132.86612194 PDPK1 3-phosphoinositide dependent 3-phosphoinositidedependent protein kinase-1 protein kinase-1 NM_014308 2.849243879P101-PI3K phosphoinositide-3-kinase, phosphoinositide-3-kinase,regulatory subunit, polypeptide regulatory subunit, polypeptide p101p101 D26120 2.833239079 splicing factor 1 X61498 2.832505941 NFKB2nuclear factor of kappa light nuclear factor of kappa light polypeptidegene enhancer in polypeptide gene enhancer in B-cells 2 (p49/p100)B-cells 2 (p49/p100) X03663 2.780905962 CSF1R colony stimulating factor1 colony stimulating factor 1 receptor, formerly McDonough receptorprecursor feline sarcoma viral (v-fms) oncogene homolog NM_0042042.756244421 PIGQ phosphatidylinositol glycan, phosphatidylinositolglycan, class class Q Q isoform 2; phosphatidylinositol glycan, class Qisoform 1 NM_003017 2.741738006 SFRS3 splicing factor, arginine/serine-splicing factor, arginine/serine- rich 3 rich 3 NM_000753 2.7408638PDE3B phosphodiesterase 3B, cGMP- phosphodiesterase 3B, cGMP- inhibitedinhibited NM_002712 2.696629047 PPP1R7 protein phosphatase 1, regulatoryprotein phosphatase 1, regulatory subunit 7 subunit 7 NM_0049062.543901205 WTAP Wilms tumor 1 associated protein Wilms' tumour1-associating protein isoform 1; Wilms' tumour 1-associating proteinisoform 2 NM_005207 2.497231245 CRKL v-crk sarcoma virus CT10 oncogenev-crk sarcoma virus CT10 oncogene homolog (avian)-like homolog(avian)-like NM_007279 2.493814707 U2AF2 U2 small nuclearribonucleoprotein U2 small nuclear ribonucleoprotein auxiliary factor(65 kD) auxiliary factor (65 kD) NM_015714 2.480620146 G0S2 putativelymphocyte G0/G1 switch putative lymphocyte G0/G1 switch gene geneNM_001511 2.476593256 CXCL1 chemokine (C-X-C motif) ligand 1 chemokine(C-X-C motif) ligand 1 (melanoma growth stimulating activity, alpha)NM_016263 2.465854406 FZR1 Fzrl protein Fzrl protein NM_0121032.459948301 AUP1 ancient ubiquitous protein 1 ancient ubiquitous protein1 isoform 1; ancient ubiquitous protein 1 isoform 2; ancient ubiquitousprotein 1 isoform 3 M32977 2.456767944 VEGF vascular endothelial growthfactor vascular endothelial growth factor NM_007040 2.432915688 E1B-AP5E1B-55 kDa-associated protein 5 E1B-55 kDa-associated protein 5 isoforma; E1B-55 kDa-associated protein 5 isoform d; E1B-55 kDa- associatedprotein 5 isoform b; E1B-55 kDa-associated protein 5 isoform c NM_0014022.404632807 EEF1A1 eukaryotic translation elongation eukaryotictranslation elongation factor 1 alpha 1 factor 1 alpha 1 NM_0023572.396934271 MAD MAX dimerization protein 1 MAX dimerization protein 1NM_005524 2.39485383 HES1 hairy and enhancer of split 1, hairy andenhancer of split 1 (Drosophila) NM_005730 2.387011766 CTDSP2 conservedgene amplified in nuclear LIM interactor-interacting osteosarcoma factor2 AF040963 2.356589317 MXD4 MAX dimerization protein 4 MAD4 M832212.351067066 RELB v-rel reticuloendotheliosis viral reticuloendotheliosisviral oncogene homolog B, nuclear factor oncogene homolog B of kappalight polypeptide gene enhancer in B-cells 3 (avian) NM_0068422.24469161 splicing factor 3b, subunit 2, 145 interleukin-1receptor-associated kDa kinase 2 NM_001570 2.239902258 IRAK2interleukin-1 receptor-associated activator of S phase kinase kinase 2NM_006716 2.230443954 ASK activator of S phase kinase rho/rac guaninenucleotide exchange factor 2 NM_004723 2.209302187 ARHGEF2 rho/racguanine nucleotide exchange human immunodeficiency virus type I factor(GEF) 2 enhancer binding protein 2 M60119 2.204995632 HIVEP2; MBP-2;human immunodeficiency virus type ras homolog gene family, member GHIV-EP2 I enhancer binding protein 2 (rho G) NM_001665 2.111301422 ARHGras homolog gene family, member G early growth response 1 (rho G) M628292.104651213 EGR1 early growth response 1 RAB11B, member RAS oncogenefamily NM_004218 2.102017164 RAB11B RAB11B, member RAS oncogene familyNM_006318 2.098191261 putative glialblastoma cell MYC-associated zincfinger protein differentiation-related U33819 2.0930233 MAZMYC-associated zinc finger protein (purine-binding transcription factor)M13150 2.069767475 MAS1 MAS1 oncogene MAS1 oncogene NM_0036852.039983511 KHSRP KH-type splicing regulatory protein KH-type splicingregulatory protein (FUSE binding protein 2) (FUSE binding protein 2)NM_006694 2.018802404 JTB; PAR; hJT; jumping translocation breakpointjumping translocation breakpoint HJTB; B PAR NM_006191 2.002365112 PA2G4proliferation-associated 2G4, 38 proliferation-associated 2G4, 38 kDakDa X78710 2.001409531 MTF1 metal-regulatory transcriptionmetal-regulatory transcription factor 1 factor 1 Gene Name PhenotypeFunction Keywords K03218 Colon cancer, advanced c-myc proto-oncogene;proto- oncogene; src gene; src oncogene NM_001201 D16431hepatoma-derived GF; hepatoma- derived growth factor X54936 placentagrowth factor M95712 Adenocarcinoma of lung, b-raf oncogene;serine/threonine somatic; Colorectal cancer, protein kinase somatic;Melanoma, melignant, somatic; Nonsmall cell lung cancer, somaticNM_005850 NM_005937 NM_001419 NM_002613 protein kinase NM_014308 D26120X61498 NF-kb subunit X03663 Myeloid malignancy, c-fms oncogene; fmsoncogene; predisposition to glycoprotein; membrane protein;proto-oncogene; signal peptide NM_004204 NM_003017 NM_000753 NM_002712regulatory polypeptide of protein phosphatase-1 NM_004906 NM_005207NM_007279 NM_015714 NM_001511 NM_016263 NM_012103 M32977 Diabeticretinopathy, angiogenic mitogen; vascular NIDDM-related, endothelialgrowth factor susceptibility to NM_007040 NM_001402 NM_002357 NM_005524NM_005730 AF040963 M83221 NF-kappa-B transcpription I-Rel; NF-kappa-Btranscription factor p50-subunit inhibitor factor inhibitor NM_006842NM_001570 NM_006716 NM_004723 M60119 NM_001665 M62829 transcriptionfactor NM_004218 NM_006318 U33819 M13150 mas oncogene; mas protein;membrane protein; proto-oncogene NM_003685 NM_006694 NM_006191 X78710metal-regulatory transcription factor; MTF-1 gene; transcription factor

TABLE 2 Immune system gene regulated by the HPLC-purified peptide Immunesystem gene enhanced by the peptide Gene Name TDP Description Product 1K02770 11.69 interleukin 1, beta interleukin 1, beta proprotein 2 J041307.341 chemokine (C—C motif) ligand 4 chemokine (C—C motif) ligand 4precursor 3 NM_000211 3.326 integrin, beta 2 (antigen CD18 (p95),integrin beta chain, beta 2 precursor lymphocyte function-associatedantigen 1; macrophage antigen 1 (mac-1) beta subunit) 4 X03663 2.781colony stimulating factor 1 receptor, colony stimulating factor 1receptor formerly McDonough feline sarcoma precursor viral (v-fms)oncogene homolog 5 NM_006186 2.06 nuclear receptor subfamily 4, group A,nuclear receptor subfamily 4, group A, member 2 member 2 isoform a;nuclear receptor subfamily 4, group A, member 2 isoform b; nuclearreceptor subfamily 4, group A, member 2 isoform c; nuclear receptorsubfamily 4, group A, member 2 isoform d 6 NM_002000 1.871 Fc fragmentof IgA, receptor for Fc alpha receptor isoform a precursor; Fc alphareceptor isoform b precursor; Fc alpha receptor isoform c precursor; Fcalpha receptor isoform d; Fc alpha receptor isoform e; Fc alpha receptorisoform f; Fc alpha receptor isoform g; Fc alpha receptor isof 7NM_003199 1.621 transcription factor 4 transcription factor 4 isoform b8 NM_001733 1.543 complement component 1, r subcomponent complementcomponent 1, r subcomponent 9 NM_002983 1.542 chemokine (C—C motif)ligand 3 chemokine (C—C motif) ligand 3 10 NM_002969 1.53mitogen-activated protein kinase 12 mitogen-activated protein kinase 1211 NM_000173 1.529 glycoprotein Ib (platelet), alpha plateletglycoprotein Ib alpha polypeptide polypeptide precursor 12 D10202 1.506platelet-activating factor receptor platelet-activating factor receptor1 NM_006864 0.662 leukocyte immunoglobulin-like receptor, leukocyteimmunoglobulin- like receptor, subfamily B (with TM and ITIM subfamily B(with TM and ITIM domains), member 3 domains), member 3 2 NM_0070520.662 NADPH oxidase 1 NADPH oxidase 1 isoform long; NADPH oxidase 1isoform short; NADPH oxidase 1 isoform long variant 3 NM_002258 0.653killer cell lectin-like receptor killer cell lectin-like receptorsubfamily B, member 1 subfamily B, member 1 4 NM_001776 0.646ectonucleoside triphosphate ectonucleoside triphosphatediphosphohydrolase 1 diphosphohydrolase 1 5 NM_016523 0.644 killer celllectin-like receptor killer cell lectin-like receptor subfamily F,member 1 subfamily F, member 1 6 NM_014442 0.641 sialic acid bindingIg-like lectin 8 sialic acid binding Ig-like lectin 8 7 NM_004133 0.635hepatocyte nuclear factor 4, gamma hepatocyte nuclear factor 4, gamma 8NM_002121 0.633 major histocompatibility complex class majorhistocompatibility complex, class II, DP beta 1 II, DP beta 1 precursor9 NM_000397 0.631 cytochrome b-245, beta polypeptide cytochrome b-245,beta polypeptide (chronic granulomatous disease) (chronic granulomatousdisease) 10 NM_004750 0.626 cytokine receptor-like factor 1 cytokinereceptor-like factor 1 11 NM_003891 0.618 protein 2, vitamin K-dependentplasma protein Z, vitamin K-dependent plasma glycoprotein glycoprotein12 NM_002260 0.603 killer cell lectin-like receptor killer celllectin-like receptor subfamily C, member 2 subfamily C, member 2 13NM_005545 0.579 immunoglobulin superfamily containing immunoglobulinsuperfamily containing leucine-rich repeat leucine-rich repeat 14NM_001311 0.561 cysteine-rich protein 1 (intestinal) cysteine-richprotein 1 (intestinal) 15 NM_004528 0.561 microsomal glutathioneS-transferase 3 microsomal glutathione S-transferase 3 16 NM_0186610.528 defensin, beta 103 defensin, beta 103, precursor 17 NM_0021240.502 major histocompatibility complex class major histocompatibilitycomplex, class II, DR beta 1 II, DR beta 1 precursor 18 Y00815 0.475protein tyrosine phosphatase, protein tyrosine phosphatase, receptortype, F receptor type, F isoform 1 precursor; protein tyrosinephosphatase, receptor type, F isoform 2 precursor 19 NM_004636 0.459sema domain, immunoglobulin domain sema domain, immunoglobulin domain(Ig), short basic domain, secreted, (Ig), short basic domain, secreted,(semaphorin) 3B (semaphorin) 3B 20 NM_001783 0.4 CD79A antigen(immunoglobulin- CD79A antigen isoform 1 precursor; associated alpha)CD79A antigen isoform 2 precursor 21 NM_004106 0.382 Fc fragment of IgE,high affinity I, Fc fragment of IgE, high affinity I, receptor for;gamma polypeptide receptor for, gamma polypeptide precursor 22 U776040.349 microsomal glutathione S-transferase 2 microsomal glutathioneS-transferase 2 23 NM_002118 0.335 major histocompatibility complex,major histocompatibility complex, class II, DM beta class II, DM betaprecursor 24 NM_002123 0.328 major histocompatibility complex, majorhistocompatibility complex, class II, DQ beta 1 class II, DQ beta 1precursor 25 K01171 0.319 major histocompatibility complex, majorhistocompatibility complex, class II, DR alpha class II, DR alphaprecursor 26 NM_006120 0.298 major histocompatibility complex, majorhistocompatibility complex, class II, DM alpha class II, DM alphaprecursor Gene Name Phenotype function Keywords 1 K02770 interleukin-1receptor binding; interleukin; interleukin 1 signal transducer activity2 J04130 receptor signaling protein act2 gene; immune activationtyrosine kinase activity; gene chemokine activity 3 NM_000211 Leukocyteadhesion cell adhesion receptor activity deficiency 4 X03663 Myeloidmalignancy, macrophage colony stimulating c-fms oncogene; fms oncogene;predisposition to factor receptor activity; ATP glycoprotein; membraneprotein; binding; transferase activity proto-oncogene; signal peptide 5NM_006186 Parkinson disease steroid hormone receptor activity;transcription factor activity 6 NM_002000 receptor activity; receptorsignaling protein activity 7 NM_003199 RNA polymerase II transcriptionfactor activity; DNA binding 8 NM_001733 Clr/Cls deficiency, complementcomponent Clr combined activity;trypsin activity; calcium ion binding;chymotrypsin activity; hydrolase activity 9 NM_002983 chemokineactivity; antiviral response protein activity; signal transduceractivity 10 NM_002969 MAP kinase activity; ATP binding; protein serine/threonine kinase activity; transferase activity; SAP kinase 3 activity11 NM_000173 Bernard-Soulier thrombin receptor activity; syndrome celladhesion molecule activity 12 D10202 platelet activating factorG-protein coupled receptor; PAF receptor activity receptor;platelet-activating factor receptor 1 NM_006864 receptor activity 2NM_007052 superoxide-generating NADPH oxidase activity; oxidoreductaseactivity; voltage-gated proton channel activity 3 NM_002258 sugarbinding; transmembrane receptor activity 4 NM_001776 apyrase activity;magnesium ion binding; hydrolase activity 5 NM_016523 transmembranereceptor activity 6 NM_014442 sugar binding; transmembrane receptoractivity; cell adhesion molecule activity 7 NM_004133 steroid hormonereceptor activity; steroid binding; transcription factor activity 8NM_002121 Beryllium disease, class II major histo- chronic,compatibility complex antigen susceptibility to 9 NM_000397 Chronicvoltage-gated ion channel granulomatous activity; electron transporterdisease, X-linked activity; oxidoreductase activity 10 NM_004750Cold-induced receptor activity sweating syndrome 11 NM_003891 trypsinactivity; protein binding; calcium ion binding; chymotrypsin activity 12NM_002260 sugar binding; transmembrane receptor activity 13 NM_005545protein binding 14 NM_001311 zinc ion binding 15 NM_004528 peroxidaseactivity; glutathione transferase activity 16 NM_018661 antimicrobialpeptide activity; Gram-positive antibacterial peptide activity 17NM_002124 Pemphigoid, MHC class II receptor activity susceptibility to18 Y00815 antigen; cell surface glycoprotein; glycoprotein;immunoglobulin superfamily; LAR gene; leukocyte common antigen; neuralcell adhesion molecule; transmembrane protein 19 NM_004636 20 NM_001783transmembrane receptor activity 21 NM_004106 receptor signaling proteinactivity; transmembrane receptor activity; IgE binding 22 U77604glutathione transferase activity; enzyme activator activity 23 NM_002118chaperone activity; MHC class II receptor activity 24 NM_002123Creutzfeldt-Jakob disease, variant, resistance to 25 K01171 MHC class IIreceptor activity antigen; class II antigen; histocompatibility antigen;major histocompatibility complex 26 NM_006120

The present inventor has verified the validity of this diagnosis methodusing 55000 cancer-related genes. When such a great number ofcancer-related genes are used, DNA chip technology is preferably used.In the DNA chip technology, each DNA of the cancer-related genes isimmobilized so as to be arrayed on a slide; the above-mentioned labeledcRNA solution is dropped into the slide; the cRNA which is nothybridized is removed by washing; and fluorescence intensity of eachgene position is detected by a scanner. The results are compared withthe result which is obtained by conducting the same process using theabove-mentioned control solution. Thus, cancer-related genes of whichexpression is increased or decreased when cells or tissues are treatedwith the peptide of the present invention can be exhaustively analyzed.

Therefore, a combination of such a cancer-related gene, the peptide ofthe present invention, and a polynucleotide encoding the peptide isuseful as a cancer diagnostic reagent kit for determining a tendency ofcanceration or malignancy of cancer.

[Diagnosis Method 3]

The peptide of the present invention is also used as an antigen forpreparing a specific antibody against the peptide. The specific antibodyis useful as a diagnostic reagent. The antibody may be either polyclonalor monoclonal antibody, and these antibodies can be prepared by knownmethods. For example, the polyclonal antibody can be prepared as anantiserum by immunizing an animal such as mouse, rat, or rabbit with thepeptide of the present invention. The antiserum may be further purified.The monoclonal antibody can be prepared by a hybridoma method:extracting spleen cells from the above-mentioned sensitized animal andfusing the spleen cells with myeloma cells to form hybridomas.

In the immunization, an appropriate adjuvant may be used. In addition,the peptide of the present invention may be used as a conjugate with acarrier protein such as keyhole limpet hemocyanin for increasing theimmunogenicity of the peptide.

The antibody against the peptide of the present invention is used as adiagnostic reagent for determining malignancy of cancer, wherein aspecimen, such as cancer tissues or cells, is stained by using thespecific antibody labeled with an appropriate fluorescence dye or thelike. When the peptide of the present invention is detected in thetissues or cells, it is suggested that cancer is advancing. The amountof the peptide can be determined by measuring the fluorescenceintensity. A large amount of the peptide indicates that the malignancyof cancer is considerably high.

Additionally, the antibody against the peptide of the present inventioncan be used as a diagnostic reagent for measuring a tendency ofcanceration (easiness of conversion to cancer: a degree of risk ofcanceration). In such a use of the antibody, a specimen of the tissuesor cells shown in diagnosis methods 1 and 2 is stained using the labeledantibody. When the peptide of the present invention is detected in thesetissues or cells, it is suggested that the tissues or cells are exposedto cancer-inducing stress, i.e., a degree of risk of canceration ishigh. When the amount of the peptide of the present invention is large,it is suggested that the tendency is further higher. When the specimenis cancer cells, a large amount of the peptide suggests that themalignancy of the cancer is high.

Furthermore, the antibody against the peptide of the present inventioncan be expected to have an anticancer effect such as suppression ofcancer cell proliferation as a cancer-cell proliferation-suppressingdrug. In addition, the antibody can be expected to block malignantalteration or mutation acceleration to which normal cells are subjectedas a cancer-preventing drug. Furthermore, by binding the antibody to ananticancer agent, a drug which may be used for missile therapy can beobtained.

EXAMPLES

Examples of the present invention will now be described, but the presentinvention is not limited to these examples.

Example 1

Preparation and Sequencing of the Peptide of the Present Invention

(Cell: UTC-8)

The squamous-cell carcinoma cell (UTC-8: FERM BP-08611) is a highlydifferentiated type with high metastasis potential and keratinizationtendency. The cell also has high adhesion and proliferation ability andbecomes confluent in 5 to 7 days after inoculating 1×10⁵ cells into aculture medium (5 ml) in a T25 culture flask (Falcon).

(Cell-culturing condition)

The following processes were performed: inoculating 1×10⁶ UTC-8 cellsinto 10 T150 culture flasks (Falcon); performing initial culture in 20ml RPMI1640 culture medium with 10% FCS (GIBCO); and additionallysupplying 30 ml of the culture medium when the proliferation reachedabout 30% of the culture flask dimension. The culture was performed in5% CO₂ and 95% humidity at an incubator internal temperature of 37° C.When the cells become 80% confluent (the proliferation of the cells ismaintained at the point just before the cell proliferation reaches itspeak), the antigen peptide bound to HLA was eluted from the cellmembrane surfaces.

(Antigen Peptide-eluting Procedure)

The culture medium for the UTC-8 cells was removed when the cells became80% confluent in each T150 culture flask (Falcon). The cells were washedwith Hanks solution once and then with 30 ml of a PBS solution notcontaining divalent calcium ion and divalent magnesium ion twice. Then,after the sufficient removal of the solution, 10 ml of acitrate-phosphate buffer having a pH of 3.3 to 3.4 was added to eachflask, and the cells were left at a room temperature for 2 min.

Then, the citrate-phosphate buffer solution was collected andcentrifuged at 1200 rpm for 7 min, and the supernatant was filteredthrough a 0.45 μm filter (Millex-HV PVDF: MILLIPORE). The filtrate wasfurther filtered through a 0.22 μm filter. The filtrate was desaltedwith Sep-Pac C18 cartridge (Waters), and the target substance bound tothe cartridge was eluted with a 60% (v/v) acetonitrile aqueous solutionas a crude extract solution. The thus obtained crude extract solutionwas frozen and stored at −20° C. for later HPLC.

The cells received the above-described treatment were washed,immediately after the collection with the citrate-phosphate buffersolution having a pH of 3.3 to 3.4, with 30 ml of Hanks solution twice.Then, the cells were recultured in 30 ml of RPMI 164 medium containing10% FCS. This treatment was subjected to the same cells once a day forsuccessive 4 days.

(Lyophilization and Redissolution of Crude Extraction)

The crude extract solution obtained in above was lyophilized using alyophilizer (FD-1000: EYELA) under conditions at a trapping temperatureof −40° C. and at a degree of vacuum of 15 Pa or less. The dried samplewas redissolved in 5 ml of the citrate-phosphate buffer solution havinga pH of 3.3 to 3.4. At this stage, the total protein amount determinedby Lowry method was 400 μg.

(Two-dimensional Electrophoresis)

Ten micrograms of the thus obtained molecule was applied totwo-dimensional electrophoresis, but no band was visible to the nakedeye by Coomassie Brilliant Blue (CBB) R250 staining (detectionsensitivity: 1 μg) and also by silver staining having a sensitivity ashigh as about 1000 times that of the CBB staining, (theoreticalsensitivity: 1 ng, actual sensitivity: about 20 times that of the CBBstaining). This suggests that the peptide molecule physically passesthrough a gel (silica gel C18) generally used in the two-dimensionalelectrophoresis. This is an unusual characteristic of the peptidemolecule of the present invention.

(Fractionating by Gel Filtration)

Since the molecule of the present invention cannot be detected by usualtwo-dimensional electrophoresis, it was tried to detect the molecule byan HPLC system. The target peptide was obtained as a fraction obtainedby using an AKTA Explorer 10 (Amersham Pharmacia Biotech) HPLC systemand Superdex Peptide 10/300 GL (Amersham Pharmacia Biotech) as agel-filtration column; using a citrate-phosphate buffer solution (0.1 Mcitric acid and 0.2 M phosphoric acid) having a pH of 6.8 as an elutionbuffer at a flow rate of 0.2 ml/min; and isolating a peak in a fractionat an elution volume of 20.8 to 22.8 ml by using a detection wavelengthof 214 nm. The obtained fraction was lyophilized and then redissolved insterilized redistilled water for desalting. The desalting was performedby fractionating by the AKTA Explorer 10 (Amersham Pharmacia Biotech)HPLC system and Superdex Peptide 10/300 GL (Amersham Pharmacia Biotech)as a gel-filtration column using distilled water as a solvent at a flowrate of 0.2 ml/min. The target peptide was obtained as a fraction at anelution volume of 11.4 to 20.0 ml.

FIG. 1 shows an elution profile of the gel filtration.

The peptide was lyophilized using the lyophilizer FD-1000 (EYELA) andstored at −20° C. for later mass spectrometry.

(Optimal pH)

The optimal pH of the elution buffer was 6.8 to 7.2. When the pH of theelution buffer was 7.3 or more, peaks were further divided into smallerpeaks. When the pH of the elution buffer was 6.7 or less, peaks, whichwere isolated from each other at a pH of 6.8, lapped over each other,and the gel filtration resolution was decreased.

(Detection Wavelength)

The peptide is characterized by the fact that the peak of the peptide inthe crude extract solution can be separately detected by usingphysiological activity as an indicator only when a detection wavelengthof 214 nm is used. However, the peptide cannot be detected when adetection wavelength of 254 to 257 nm or of 280 nm, which are generallyused in peptide detection, is used. These results suggest that the aminoacid composition of the peptide has extremely low contents of tyrosine(absorption wavelength: 280 nm), tryptophan (absorption wavelength: 280nm), and phenylalanine (absorption wavelength: 257 nm); or that thepeptide does not substantially contain such amino acids. The use of adetection wavelength of 214 nm has a demerit such that many interferingsubstances are also detected by ultraviolet absorption spectrometry.However, it can precisely measure a protein amount (quantitativedetection range: 5 to 1000 μg) regardless of types of proteins. The 214nm is the most appropriate wavelength for, as in this experiment,precisely measuring a concentration of unknown peptide in a crudeextract solution by detecting a peak utilizing peptide-bond absorptionwithout denaturation of the peptide to maintain the physiologicalactivity. On the other hand, the quantitative detection property wasfurther improved by using a detection wavelength of 205 nm when thecrude extract solution containing the peptide was applied to a desaltingcolumn (Sep-Pack 18) or to a molecular sieve (Centricon YM-3) forremoving foreign substances such as nucleic acids as far as possible, byintending only purification of the peptide without any concern formaintaining the physiological activity. Therefore, when a peak in thecrude extract solution was detected using the physiological activity asan indicator, a detection wavelength of 214 nm was used, and when theprecise quantitative determination was necessary, a detection wavelengthof 205 nm was used.

(Fractionating Flow Rate in Column)

The fractionating flow rate is important for separating the peptide.Namely, the flow rate of an elution buffer is determined so thatmolecules slowly pass through the column to effectively utilizeadvantages of the molecular sieve. When the flow rate was 0.28 ml/min,adjacent peaks lapped over each other; thus, the separation resolutionwas insufficient. For the detection of the peak of the presentinvention, a low flow rate of 0.14 ml/min was optimal. Each fraction wasdetermined to be 0.1 ml/well from the view point of the width of thepeak, and was collected in a 96-well plate (Nunc).

(Structure Analysis of the Peptide by Mass Spectrometer)

The peptide fraction that was stored after lyophilization wasredissolved in sterilized redistilled water, and 5 μg as a protein wasseparated by ProteinChip Series 4000 system (Ciphergen). The sample (5μg) was bound to the ProteinChip by using MilliQ water as a binding/washbuffer in normal-phase ProteinChip and using 100 mM sodium acetate (pH4.0) as a binding/wash buffer in cation exchange ProteinChip, and wasmeasured by using alpha-cyano-4-hydroxy-cinnamic acid (CHCA) as anenergy-absorbing molecular in a mass range of 800 to 2500 m/z. As aresult, a peak of a molecular weight which was thought to be that of thetarget peptide was detected. By using the detection conditions clarifiedby the above-described processes as a reference, the target peptidetrapped on the chip was analyzed by a mass spectrometer, QSTAR XLLC/MC/MS system (Applied Biosystems). Then, on the basis of the resultsof time-of-flight (TOF mass spectrometry) analysis of the fragmentedpeptide-constituting portion, a data base (Mascot Search Results: MatrixScience) was searched for a sequence of the peptide to reveal that thepeptide had the following peptide sequence of which function was unknownas of Jan. 27, 2005. Peptide: Gln-Pro-Gln-Phe-Gly-Arg-Arg-Met-Glu- (SEQID NO: 1) Ser-Lys

Example 2

Confirmation of Expression of the Peptide of the Present Invention inOriginal Cancer Cell UTC-8

The fact that the peptide obtained in Example 1 was actually expressedin the original cancer cell UTC-8 at a gene level was confirmed asfollows:

UTC-8 cells after the extraction of the peptide of the present inventionwere recultured, and total RNA was extracted from the recultured UTC-8cells after 2 hr, 4 hr, and 6 hr from the extraction, respectively, byusing an RNA extraction kit (QIAGEN). Then, cDNA was synthesized usingeach of the extracted total RNA as a template to produce a cDNA pool. Onthe basis of the amino acid sequence (SEQ ID NO: 1) of the peptide, thefollowing primers were designed. Fw primers (DNAs encoding fourcontiguous amino acids of the peptide were synthesized so that theN-terminal of the four contiguous amino acids shifted one by one fromthe N-terminal of the peptide toward the C-terminal.) MHC 1-4 CAR CCNCAR TTY (SEQ ID NO: 2) MHC 2-5 CCN CAR TTY GGN (SEQ ID NO: 3) MHC 3-6CAR TTY GGN AGR (SEQ ID NO: 4) MHC 4-7 TTY GGN AGR AGR (SEQ ID NO: 5)MHC 5-8 GGN AGR AGR ATG (SEQ ID NO: 6) MHC 6-9 AGR AGR ATG GAR (SEQ IDNO: 7) MHC 7-10 AGR ATG GAR TCN (SEQ ID NO: 8) MHC 8-11 ATG GAR TCN AAR(SEQ ID NO: 9)(N: A, T, C, or G, R: G or A, Y: C or T) Rv primer (a gene sequenceproduced for an amino acid sequence at the C-terminal of the peptide):Oligo-dT primer (poly-A primer)

Here, the poly-A primer was used as the Rv primer in order to obtaincDNA synthesized from mRNA.

Complementary DNAs were synthesized by reverse transcription from totalRNA which was extracted at each time to produce a cDNA pool. Then, PCRwas performed using this cDNA pool as templates and using primerssynthesized as described above under conditions at 94° C. for 3 min, at55° C. for 1 min, and at 74° C. for 1 min as one cycle. The cycle wasrepeated 35 cycles in total. After agarose-gel electrophoresis, 12 bandswere selected from a sample of which RNA was extracted after 4 hr whenthe signal intensity was largest. The size of each band was about 500 to2000 bp. Genes of these bands extracted from the gel were inserted intopGM easy vectors.

Insert of the genes into 12 types of the pGM easy vectors was checked,and 8 samples per one type of the vector were sequenced. As a result, itwas confirmed that all the sequences of the 8 samples included a commonsequence shown in Table 3 below, though the DNA lengths of the sequenceswere different from those of each other. TABLE 3

The result shows the fact that a gene being common in or complementaryto the gene encoding the peptide of the present invention is surelypresent in original cancer cell UTC-8 and that the peptide is derivedfrom UTC-8 cells.

Example 3

Artificial Synthesis of the Peptide of the Present Invention by FmocMethod

The peptide was artificially synthesized by an in vitro synthesis system(Abacus: Sigma Genosys).

The peptide was synthesized using an activated cellulose membrane(SPOTs: Sigma Genosys) as follows: The α-amino groups of amino acidswere protected with 9-fluorenyl methoxy carbonyl (Fmoc) and the carboxylgroups were protected with an active ester (Opfp or Odhbt). These aminoacid derivatives were dissolved in 1-methyl-2-pyrrolidinone (NMP). About1 μl of the amino acid derivative solution (equivalent to 100 to 200 μgof Fmoc-amino acid) was spotted on the membrane at each marked positionwhere a spacer arm was bound thereto. The end of the spacer arm had afree amino group, and the spots were colored to blue with bromophenolblue (BPB). An amido bond was formed by the reaction between the aminogroup on the membrane and the active ester. Then, capping was carriedout as follows: Excess amino acid derivatives were rinsed away withdimethylformamide (DMF), and then unreacted amino group was acetylatedby treating with acetic anhydride/DMF so as to be lost the reactivity.Then, the Fmoc group protecting the amino group was removed by secondaryamine, piperidine/DMF, in order to let the amino group free for thesubsequent extension reaction. Additionally, after all synthesisprocesses were completed, deprotection of the side chains was carriedout. Namely, a t-butyl alcohol-based protecting group (Pmc, OtBu, Trt,tBoc, tBu, etc.) protecting the reactive side chains was removed byusing trifluoroacetate (TFA) mixed with dichloromethane(DCM)/triisobutylsilane. Thus, the target peptide was artificiallysynthesized.

Example 4

Influence of the Artificially Synthesized Peptide on Normal Cell Gene

(a) The peptide obtained in Example 3 was added to a human normalperipheral blood monocyte culture system (10 to 100 μg peptide/1×10⁶ to1×10⁷ monocytes), and they were cocultured for 7 hr. Then, mRNA wasextracted from the monocytes and was applied to analysis forup-regulation and down-regulation in respect to about 55000 human genesby using DNA chip system (Clontech). Table 4 shows a part of the genes.TABLE 4 A. Enhanced Gene Expression Amount Expression Amount Gene beforeTreatment after Treatment with No. Gene Name Gene Bank with the Peptidethe Peptide 1. Oncogene 1 21713 NM_004339.2 GE480864 DISCOVERY0.168670654 7.741943359 2 32050 CB999164.1 GE540894 DISCOVERY0.344818115 9.805541992 3 34854 NM_198502.1 GE603511 DISCOVERY0.040802002 1.098205566 4 46997 AL831827.1 GE547122 DISCOVERY11.17044067 236.1818237 5 37781 AI478531.1 GE645872 DISCOVERY2.533325195 32.40939331 6 7394 AA021565.1 GE707587 DISCOVERY 29.597229350.270813 7 22388 CD244420.1 GE692174 DISCOVERY 60.32098389 571.23254398 6231 BF973345.1 GE54360 DISCOVERY 0.583343506 5.453491211 9 34979BC041456.1 GE631189 DISCOVERY 2.651672363 24.171875 10 4210 NM_152577.1GE493259 DISCOVERY 61.90475464 393.7416382 11 36631 AW302705.1 GE53805DISCOVERY 71.11248779 417.5 12 29317 AA60U91.1 GE551636 DISCOVERY2.891571045 10.05682373 13 14195 NM_002170.2 GE55306 DISCOVERY2.811950684 7.21875 14 2183 AA861218.1 GE80525 DISCOVERY 3.4086914068.622650146 15 22615 BX117642.1 GE516913 DISCOVERY 81.63721924204.4117737 16 3858 AI263196.1 GE59314 DISCOVERY 9.184204102 22.6153869617 8431 NM_021081.3 GE56485 DISCOVERY 7.641021729 18.609375 18 18829NM_002958.1 GE81500 DISCOVERY 6.883331299 15.3151959 19 8440 NM_016232.4GE79419 DISCOVERY 7.225799561 15.71429443 20 3642 AW594132.1 GE543964DISCOVERY 1627.052002 3306.942871 21 38566 NM_031273.1 GEB4875 DISCOVERY7.339630127 14.54998779 22 24910 NM_018556.2 GE60436 DISCOVERY29.01333618 55.05334473 23 6329 BX116538.1 GE58014 DISCOVERY 37.8082275467.53659058 2. Immune System Gene 1 25767 INCYTE UNIQUE GE59636DISCOVERY 130.6451721 4927.383301 2 29703 BX103139.1 GE59980 DISCOVERY0.660003662 19.8302002 3 28615 AA504638.1 GE520093 DISCOVERY 2.73913574220.69332886 4 30707 AI809890.1 GE54001 DISCOVERY 2.853668213 21.056610115 21607 NM_002960.1 GE80951 DISCOVERY 3.446289063 24.92727661 6 51904H08511.1 GE893705 DISCOVERY 2.304870605 11.3684082 7 22254 NM_176891.2GE80659 DISCOVERY 26.98571777 122.6947327 8 45170 BM671692.1 GE53155DISCOVERY 38.31506348 85.25881958 9 22100 BX457477.2 GE616415 DISCOVERY328.440918 608.4909058 A. Enhanced Gene Enhancement Ratio Description 1.Oncogene 1  45.89976479 zn87blly5 Stratagene lung carcinoma 937218 cDNAclone IMAGE: 565149 5′ similar to contains Alu repetitive element;contains element MER22 repetitive element; 2  28.43685282 disrupted inrenal carcinoma 1 (DIRC1) 3  26.91548242 Homo sapiens NEUROBLASTOMA COT25-NORMALIZED cDNA clone CS0DC018YE24 3-PRIME 4  21.14346521 clone N11NTera2D1 teratocarcinoma mRNA 5  12.79322267 NEUROBLASTOMA COT25-NORMALIZED cDNA clone CS0DC011YD03 5-PRIME 6  11.8345813 HELA CELLSCOT 25-NORMALIZED cDNA clone CS0K007YM05 3-PRIME 1  9.469881078zb91f05s1 Soares_parathyroid_tumor_NbHPA cDNA clone IMAGE: 320193 3′ 8 9.348679048 breast carcinoma amplified sequence 1 (BCAS1) 9 9.115709518 yu38e06r1 Soares ovary tumor NbHOT cDNA clone IMAGE: 2360985′ 10  6.360442594 RAS-like, family 11, member A (RASL11A) 11 5.87098009 RAB43, member RAS oncogene family (RAB43), mRNA 12 3.477979124 T-cell leukemia/lymphoma 6 (TCL6), transcript variantTCL6a1 13  2.567168067 B-cell translocation gene 4 (BTG4) 14 2.529607148 neuro-oncological ventral antigen 1 (NOVA1), transcriptvariant 1 15  2.497784939 endogenous retroviral family W, env(C7),member 1 (syncytin) (ERVWE1), mRNA 16  2.46242208 preferentiallyexpressed antigen in melanoma (PRAME), transcript variant 3 17 2.435456365 Ras-associated protein Rap1 (RBJ) 18  2.225055752 v-mycmyelocytomatosis viral related oncogene, neuroblastoma derived (avian)(MYCN) 19  2.174748179 mab-21-like 2 (C elegans) (MAB21L2) 20 2.032475217 RAB2, member RAS oncogene family (RAB2) 21  1.982387061 RASprotein activator like 2, mRNA (cDNA clone IMAGE: 5399841), withapparent retained intron 22  1.891518589 platelet-derived growth factorbeta polypeptide (simian sarcoma viral (v-sis) oncogene homolog)(PDGFB), transcript variant 2 23  1.186293486 mutated in colorectalcancers (MCC) 2. Immune System Gene 1  37.71577029 tumor necrosis factor(TNF superfamily, member 2) (TNF) 2  30.04559116 chemokine (C-X-C motif)ligand 9 (CXCL9) 3  7.554692722 IL3-UT0117-080301-496-D07 UT0117 Homosapiens cDNA 4  7.378787069 immunoglobulin superfamily, member 4C(IGSF4C) 5  7.233077713 colony stimulating factor 1 (macrophage) (CSF1),transcript variant 1 6  4.932341181 interleukin 1 family, member 8 (eta)(IL1F8), transcript variant 1 7  4.546654408 interleukin 4 (IL4),transcript variant 1 8  2.225203663 transforming growth factor beta 1induced transcript 1 (TGFB1I1) 9  1.852664734 IL5-CI0001-181000-178-f03CI0001 Homo sapiens cDNA B. Suppressed Gene Expression Amount ExpressionAmount Gene before Treatment after Treatment with No. Gene Name GeneBank with the Peptide the Peptide 1. Cancer- Suppressing Gene 1 35687BX108016.1 GE82100 DISCOVERY 103.9224243 0.225219727 2 36334 BF433017.1GE82101 DISCOVERY 341.440918 2.079650879 3 37600 AW979088.1 GE62125DISCOVERY 982.9432373 8.42855835 4 30909 NM_018412.2 GE79080 DISCOVERY987.7108154 11.47369385 5 8280 BC063301.1 GE62103 DISCOVERY 758.41540539 6 11436 NM_152271.2 GE82056 DISCOVERY 1207.091431 14.74417114 7 11261NM_018559.2 GE56225 DISCOVERY 379.1338501 6.257141113 8 50395NM_004480.3 GE79552 DISCOVERY 470.2086792 8.763153076 9 52111 AK056875.1GE61359 DISCOVERY 593.6870728 13.6000061 2. Immune System Gene 1 20881NM_001482.1 GE61110 DISCOVERY 6599.050293 38.16882324 2 47973NM_004394.1 GE81458 DISCOVERY 1480.68396 9.638885498 3 10791 W00901.1GE80378 DISCOVERY 517.4453125 3.42855835 4 15907 BU899259.1 GE80960DISCOVERY 912.8641357 6.363647461 5 16756 AI707455.1 GE81522 DISCOVERY395.0359497 2.80645752 6 34736 AV736303.1 GE80100 DISCOVERY 3974.22119129.0786438 7 39991 AI252940.1 GE61199 DISCOVERY 2414.748779 17.774200448 41726 AI453596.1 GE58507 DISCOVERY 4512.05127 33.50683594 9 45767BG190549.1 GE79374 DISCOVERY 2359.263184 18.125 10 42614 AW296107.1GE81744 DISCOVERY 626.2438965 4.848480225 11 20551 AK024566.1 GE86210DISCOVERY 2811.726807 22.93103027 12 38103 NM_173695.1 GE61298 DISCOVERY703.8875122 5.777770996 13 37068 AW971488.1 GE58019 DISCOVERY6960.161133 57.38461304 14 4899 BX115183.1 GE80961 DISCOVERY 536.46795654.5 15 43032 BC013284.2 GE58028 DISCOVERY 519.3710938 4.445770264 1642813 NM_001763.1 GE58393 DISCOVERY 4447.713379 38.51351929 17 22326NM_014396.2 GE82907 DISCOVERY 1102.541626 9.742858887 18 37445NM_007068.2 GE60016 DISCOVERY 1671.436768 14.875 19 22890 BX119833.1GE86305 DISCOVERY 1112.63855 9.902160645 20 16478 NM_020530.3 GE54528DISCOVERY 663.3167725 6.018188477 21 52539 AI133415.1 GE82473 DISCOVERY17346.24609 158.6896667 22 41803 NM_178313.1 GE735470 DISCOVERY89.23596191 0.820007324 23 41817 NM_016292.1 GE79992 DISCOVERY10618.77539 98.42697144 24 18884 NM_015604.2 GE57715 DISCOVERY339.244873 3.387084961 25 8922 NM_024077.2 GE57504 DISCOVERY 10964.4248110.8526306 26 24969 AV752332.1 GE60370 DISCOVERY 2446.13867225.71212769 27 8634 BC043004.2 GE566269 DISCOVERY 947.6875 10.1956481928 38879 NM_182832.1 GE476899 DISCOVERY 2933.067871 31.93505859 29 49586BG289120.1 GE58687 DISCOVERY 248.4822998 2.715911865 30 11651 AW850450.1GE85351 DISCOVERY 1144.841431 12.83999634 31 53172 NM_022749.4 GE59644DISCOVERY 577.4226074 6.560333252 32 49622 CB052158.1 GE59962 DISCOVERY3421.524414 39.68115234 33 22868 NM_152285.1 GE79323 DISCOVERY539.8291016 6.525421143 34 23827 BG221408.1 GE59882 DISCOVERY 2403.9887729.7802124 35 18139 AA938869.1 GE890404 DISCOVERY 2660.28491233.11999512 36 22789 BX104097.1 GE61439 DISCOVERY 731.80696 9.72549438537 11236 NM_003937.1 GE58794 DISCOVERY 4738.49707 65.4675293 38 10204AW197778.1 GE81039 DISCOVERY 696 9.714294434 39 49170 AA577911.1 GE61247DISCOVERY 713.4000244 10.09091187 40 42474 AW137161.1 GE62833 DISCOVERY1164.558472 17.03775024 41 52342 NM_006147.2 GE54992 DISCOVERY1200.007446 18.71737671 42 42573 NM_022648.2 GE79383 DISCOVERY953.4078369 15.75 43 24440 NM_182936.1 GE592149 DISCOVERY 14 0.23477172945 24440 NM_182936.1 GE592149 DISCOVERY 14 0.234771729 46 24440NM_182936.1 GE592149 DISCOVERY 14 0.234771729 47 46186 NM_032746.1GE82057 DISCOVERY 1407.181763 25.55319214 48 39926 INCYTE UNIQUE GE82589DISCOVERY 17409.49609 317.7109375 49 47689 AK097380.1 GE61992 DISCOVERY3803.877441 72.08435059 50 12812 CF135919.1 GE55506 DISCOVERY 1176.7252224.53225708 51 15994 R25284.1 GE79364 DISCOVERY 11397.33301 239.473693852 52071 BM698907.1 GE86827 DISCOVERY 415.7030029 9.42855835 53 40727BG573885.1 GE55241 DISCOVERY 1204.090942 28.16665649 54 11635 AI376607.1GE80662 DISCOVERY 106.1363525 2.5 55 4403 N26032.1 GE56202 DISCOVERY891.694458 21.96551514 56 14949 BX110547.1 GE81738 DISCOVERY 1020.04296925.26760864 57 51004 BM670853.1 GE59115 DISCOVERY 345.97058118.725006104 58 17177 AI274757.1 GE54607 DISCOVERY 1033.54187 26.1604919459 9938 NM_152288.1 GE87537 DISCOVERY 11.78378296 0.299987793 60 21487NM_006861.4 GE58813 DISCOVERY 387.6266479 9.909088135 61 37834BU608350.1 GE57222 DISCOVERY 545.7602539 14.10638428 62 42661 C04533.1GE60353 DISCOVERY 1506.339355 39.14474487 63 42680 AW449970.1 GE80314DISCOVERY 1616.487305 42.02856445 B. Suppressed Gene Suppression RatioDescription 1. Cancer- Suppressing Gene 1 461.4268293 heat shock protein75 (TRAP1) 2 164.1818449 heat shock 70 kDa protein 14 (HSPA14) 3116.6205651 AHA1, activator of heat shock 90 kDa protein ATPase homolog1 (yeast) (AHSA1) 4  86.08481528 tumor rejection antigen (gp96) 1 (TRA1)5  84.26837836 brain specific protein (CGI-38) 6  81.86905991serologically defined breast cancer antigen 84 (SDBCAG84), transcriptvariant 1 7  60.59218471 breast cancer metastasis suppressor 1 (BRMS1) 8 53.65747638 Ras suppressor protein 1 (RSU1), transcript variant 1 9 43.65344164 leukemia cell normalized cDNA library cDNA cloneLEU1757_26_C2 2. Immune System Gene 1 172.891112 natural killer celltranscript 4 (NK4) 2 153.615681 CDS antigen, beta polypeptide 1 (p37)(CD8B1), transcript variant 5 3 150.9221252 HLA-B associated transcript5 (BAT5) 4 143.4498283 intercellular adhesion molecule 2 (ICAM2) 5140.7596399 major histocompatibility complex, class I, E (HLA-E) 6136.6714768 adhesion molecule AMICA (AMICA) 7 135.8569567 lymphotoxinbeta (TNF superfamily, member 3) (LTB), transcript variant 2 8134.660619 apoptosis-associated speck-like protein containing a CARD(ASC), transcript variant 1 9 130.1662446 HLA-B associated transcript 1(BAT1), transcript variant 1 10 129.1629268 killer cell lectin-likereceptor subfamily K, member 1 (KLRK1) 11 122.6166802 IL2-inducibleT-cell kinase (ITK) 12 121.8268278 tumor necrosis factor receptorsuperfamily, member 7 (TNFRSF7) 13 121.2896762 immediate early response2 (IER2) 14 119.2151015 interferon (alpha, beta and omega) receptor 2(IFNAR2), transcript variant 1 15 116.8236465 interferon,gamma-inducible protein 16 (IFI16) 16 115.4844704 granzyme A (granzyme1, cytotoxic T-lymphocyte- associated serine esterase 3) (GZMA) 17113.1640763 immune associated nucleotide 2 (hIAN2) 18 112.365497 mal,T-cell differentiation protein (MAL), transcript variant d 19112.3632094 NK inhibitory receptor precursor (NKIR) 20 110.2186771 tumornecrosis factor (ligand) superfamily, member 12 (TNFSF12), transcriptvariant 1, mRNA 21 109.3092351 major histocompatibility complex, classII, DR alpha (HLA-DRA) 22 108.8233718 programmed cell death 2 (PDCD2),transcript variant 2 23 107.8848128 major histocompatibility complex,class I, F (HLA-F) 24 100.1583595 (clone 38-1) MHC class I mRNAfragment. 25  98.90991981 interferon induced transmembrane protein 1(9-27) (IFITM1) 26  95.13559911 CD69 antigen (p60, early T-cellactivation antigen) (CD69) 27  92.9501962 perforin 1 (pore formingprotein) (PRF1) 28  91.84476247 interferon stimulated gene 20 kDa(ISG20) 29  91.49129726 interleukin-1 receptor-associated kinase 4(IRAK4) 30  89.16213062 implantation-associated protein (DKFZp564K142)31  88.01726761 transforming growth factor, beta 1 (Camurati- Engelmanndisease) (TGFB1) 32  86.22542976 intercellular adhesion molecule 3(ICAM3) 33  82.72708991 lymphocyte cytosolic protein 2 (SH2 domaincontaining leukocyte protein of 76 kDa) (LCP2) 34  80.72436613 majorhistocompatibility complex, class II, DM alpha (HLA-DMA) 35  80.32262392major histocompatibility complex, class II, DQ alpha 1 (HLA-DQA1) 36 75.24645339 CASP8 and FADD-like apoptosis regulator (CFLAR) 37 72.37934777 natural killer cell group 7 sequence (NKG7), mRNA 38 71.64699451 interferon-related developmental regulator 1 (IFRD1) 39 70.69728028 interferon gamma receptor 2 (interferon gamma transducer 1)(IFNGR2) 40  68.35165764 programmed cell death 6 (PDCD6) 41  64.11194608apoptosis-related protein PNAS-1 (FLJ39616) 42  60.53383092 T-cellactivation protein (PGR1) 43  59.63239308 THYMUS cDNA cloneCS0CAP007YL06 5-PRIME 45  59.63239308 THYMUS cDNA clone CS0CAP007YL065-PRIME 46  59.63239308 THYMUS cDNA clone CS0CAP007YL06 5-PRIME 47 55.06872703 cell death-regulatory protein GRIM19 (GRIM19) 48 54.79665331 thymosin-like 6 (TMSL6) 49  52.7698094 killer celllectin-like receptor subfamily B, member 1 (KLRB1) 50  47.96644744ovarian carcinoma immunoreactive antigen (OCIA) 51  47.5932568beta-2-microglobulin (B2M) 52  44.08977359 interferon (alpha, beta andomega) receptor 1 (IFNAR1) 53  42.74880629 linker for activation of Tcells (LAT) 54  42.45454102 thymostn, beta 4, Y-linked (TMSB4Y) 55 40.595199 programmed cell death 4 (neoplastic transformation inhibitor)(PDCD4), transcript variant 2 56  40.36958872 programmed cell death 10(PDCD10), transcript variant 1 57  39.65276092 tumor necrosis factor(ligand) superfamily, member 10 (TNFSF10) 58  39.50773832 tumor necrosisfactor superfamily, member 5-induced protein 1 (TNFSF5IP1) 59 39.28087487 interleukin 17D (IL17D) 60  39.11829653 programmed celldeath 2 (PDCD2), transcript variant 1 61  38.68888322 proteasome(prosome, macropain) 26S subunit, non- ATPase, 1 (PSMD1) 62  38.4812664proteasome (prosome, macropain) subunit, beta type, 9 (largemultifunctional protease 2) (PSMB9), transcript variant 1 63 38.46163498 proteasome (prosome, macropain) 26S subunit, ATPase, 4(PSMC4), transcript variant 1

This experiment revealed the facts described below, and it was clarifiedthat the artificially synthesized peptide also significantly influencedthe expression of human cancer-related gene to induce cancer, as in thepeptide derived from the original cell line.

A. Enhanced Gene

1. Oncogene

Expression of oncogenes of cranial neuroblastoma, lung cancer, parotidgland tumor, breast cancer, colon cancer, renal cancer, ovarian cancer,melanoma, T-cell leukemia, B-cell leukemia, and so on were enhanced inthe range of 47 to 1.7 times the baseline expression thereof. Expressionof carcinogenic genes such as RAS, RAB, v-myc, and mab were alsoenhanced in the range of 6.7 to 1.7 times the baseline expressionthereof. Additionally, expression of cell growth factor such as aplatelet-derived growth factor was enhanced.

2. Immune System Gene

Expression of TNF, which is an inflammatory factor, was not enhanced,but expression of most of important factors as an operating factor forthe humoral immunity mechanism, which is the opposite side of anti-tumorimmunity, was enhanced.

B. Suppressed Gene

1. Cancer-suppressing Gene

Every cancer-suppressing gene was significantly suppressed: Expressionof heat shock protein relating to cancer antigen-induction wassuppressed in the range of 1/461 to 1/116 of the baseline expressionthereof, and expression of a tumor rejection antigen was suppressed inthe range of 1/86 to 1/81 of the baseline expression thereof. Expressionof a gene relating to inhibition of metastasis of breast cancer wassuppressed to 1/60, expression of a gene relating to inhibition ofleukemia was suppressed to 1/43, and expression of RAS suppressorprotein gene was suppressed to 1/43.

2. Immune System Gene

The expression of genes relating to anti-tumor immunity was highlysuppressed as follows:

suppression of a gene relating to construction of thymus tissues orproduction of a thymic hormone;

suppression of a function and a construction factor of T-cell which isimportant in inhibition of cancer;

suppression of a function and construction of NK-cell;

suppression of production of a factor for killer-cell-secretion;

suppression of an apoptosis factor relating to cellular suicide;

suppression of a factor relating to cell adhesion in cell apoptosis;

suppression of production of antineoplastic interferon; and

suppression of expression of a cell organelle, an enzyme system, and amajor histocompatibility complex (MHC) relating to induction of a cancerantigen.

(b) The peptide obtained in Example 3 was added to a culture medium (10to 100 μg peptide/1×10⁷ monocytes) for human normal peripheral bloodmonocytes extracted from subjects shown in Table 5 below, and they werecocultured for 7 hr. Then, total mRNA was extracted from the monocytes,and the amount of the total mRNA was measured and compared to that ofthe total mRNA before the treatment with the peptide. Table 5 shows theresults. TABLE 5 Effect of the artificially synthesized peptide on totalmRNA amount of peripheral blood monocytes pre post total total RNA RNApost/pre NAME (μg/ml) (μg/ml) (%) reduction ratio (%) H. S. 45.875 0.9582 98 Healthy subject (smoker) M. S. 20.779 9.188 44.2 55.8 Healthysubject (smoker) E.. E. 69.622 7.735 11.1 88.9 Healthy subject(side-stream smoke) S. G. 61.145 16.562 27 73 Healthy subject H. T.66.804 14.742 22 78 Healthy subject (side-stream smoke) M. O. 58.07619.551 33.6 66.4 Healthy subject (side-stream smoke) M. A. 52.318 17.68133.7 66.3 Healthy subject K. K. 8.078 1.757 21.7 78.3 Cancer patientreceived chemotherapy and radio- therapy* Samples are equivalent to 1 × 10⁷ peripheral blood monocytes.* Treatment time with the peptide: 7 hr* The processes for collecting total RNA are the same as those inExample 4.

With referred to the results shown in Table 5, the total mRNA in H.S.and E.E. were significantly decreased. H.S. was an addicted smoker whohas smoked for more than ten years. With respect to M.S., the reductionratio of total mRNA was low, but the pre-total mRNA amount itself wasalso low, which has been caused by smoking over a long period of time.E.E. was not a smoker, but father of E.E. was an addicted smoker.Therefore, it was assumed that the. decrease in the total mRNA wascaused by passive smoking over a long period of time. Both H.T. and M.O.were passive smokers, and the reactivity to the peptide was acceleratedin proportion to degree of the passive smoking (H.T.>M.O.). In K.K., thereduction ratio was low and the pre-total mRNA amount was significantlysmall. This subject was a cancer patient who has already received ananticancer agent and irradiation at the maximum possible dose.Therefore, the general gene translation was significantly impaired;which caused the result entirely different from that in healthysubjects. The results shown in Table 5 show that the cancer diagnosiscan be efficiently performed by analyzing the changing ratio of totalmRNA amounts before and after the treatment with the peptide of thepresent invention.

Example 5

Detection of the Peptide of the Present Invention in Cancer Cells andInfluence of the Peptide on Normal Cells

In order to examine influences at a gene level of the peptide on normalcells, RT-PCR was performed by adding the peptide artificiallysynthesized in Example 3 to the normal cells.

(Method and Result)

(1) Normal cells were treated with the artificially synthesized peptidefor 7 hr as follows: Peptide No. of Cells Amount Kidney mesangium cell:(ACBRI-1376; 1.25 × 10⁶ 12.5 μg Applied Cell Biology Research Institute)Skin fibroblast cell: (Catlog2F0-C25;  4.0 × 10⁶ 40.0 μg Cell Systems)Pancreatic epithelial cells: (ACBRI515; 1.05 × 10⁷ 105.0 μg  AppliedCell Biology Research Institute) Peripheral blood monocytes: H.S. 1.25 ×10⁷ 12.5 μg M.S. 1.20 × 10⁷ 12.0 μg E.E. 1.40 × 10⁷ 14.0 μg(Kidney mesangium cells, skin fibroblast cells, and pancreaticepithelial cells: 100 μg peptide/1.0×10⁷ cells, peripheral bloodmonocytes: 10 μg peptide/1.0×10⁷ cells)

Then, total RNA was extracted from the above-treated normal cells,untreated normal cells, and squamous-cell carcinoma cells by using anRNA extraction kit (QIAGEN). The numbers of the treated cells and theuntreated cells were the same. RNA No. of Cells Amount Kidney mesangiumcell: (ACBRI-1376; Applied 1.25 × 10⁶ 30 μg Cell Biology ResearchInstitute) Skin fibroblast cell: (Catlog2F0-C25; Cell  4.0 × 10⁶ 30 μgSystems) Pancreatic epithelial cells: (ACBRI515; 1.05 × 10⁷ 30 μgApplied Cell Biology Research Institute) Peripheral blood monocytes:H.S. 1.25 × 10⁷ 60 μg M.S. 1.20 × 10⁷ 60 μg E.E. 1.40 × 10⁷ 60 μgCervical carcinoma cells (HeLa): 1.60 × 10⁷ 60 μg

Reverse transcription was performed by using 30 μg of each extractedtotal RNA to generate cDNA pools.

PCR was conducted using each of the cDNA pools as templates.

The condition for the PCR was as follows:

PCR buffer: 6 μl

dNTP: 2 μl

Primer Fw (11 amino acids or 7 amino acids): 1 μl

Primer Rv (oligo dT): 1 μl

dH2O: 9.5 μl

taq: 0.5 μl

Sample (RT products): 10 μl

(Total: 30 μl)

PCR: 45 cycles

Initial denaturation: at 94° C. for 5 min

Denaturation: at 94° C. for 30 sec

Annealing: at 55° C. for 30 sec

Extension: at 72° C. for 1 min

(2) Each of the PCR products was applied to electrophoresis and detectedby using a fluorescence label.

(a) FIGS. 2A and 2B show the results when the full-length peptide (11amino acids) of the present invention was used as the primer in the PCR.

The results are as follows:

(i) Lanes 2 and 3 in FIG. 2A show the results of original UTC-8 cellsamples which were not treated with the peptide of the presentinvention. The original UTC-8 cell samples stably expressed the gene ofthe preset invention.

Lane 4 in FIG. 2A shows the result of a cervical carcinoma cell sample,wherein a smear was observed. This means that the sample includesvarious sizes of genes which are complementary to the gene encoding thepeptide of the present invention. Namely, the expression pattern of thegene was significantly changed in the cervical carcinoma cell sample bythe treatment with the peptide of the present invention. Therefore,samples showing such a reaction pattern are thought to have a tendencyof canceration.

(ii) On the basis of the results above, samples on each lane wereexamined. In some samples (Lanes 5 and 6 of FIG. 2A, lanes 4 and 5,lanes 6 and 7, and lanes 8 and 9 in FIG. 2B), the original stable genepattern was changed to a pattern having a broadened smear by thetreatment with the peptide of the present invention. Such samples weredetermined to have a tendency of canceration. Actually, the blood sampleof the lanes 5 and 6 of FIG. 2A was obtained from a young man who hadsmoked for more than 10 years. Though the samples were normal cells,they had a high sensitivity to the peptide. It is thought that such ahigh sensitivity is caused by that these cells were, as in the peptideof the present invention, extracted from epithelial cells and werederived from a tissue in an early stage. Therefore, the gene expressionis not fixedly stable, and the unstable gene expression is involved inthe acuity sensitivity against a highly disturbing factor from theoutside such as the peptide of the present invention.

(iii) On the contrary, in the sample of lanes 2 and 3 of FIG. 2B, genemutation was hardly induced even if the sample was treated with thepeptide of the present invention. This sample is thought to have acharacteristic to rarely get cancer. Actually, the sample was obtainedfrom a healthy young man in twenties.

(b) FIGS. 3A and 3B show the results when a DNA encoding an amino acidsequence (7 amino acids) at positions 1-7 of the peptide of the presentinvention was used as a primer in the PCR.

The results are as follows:

(i) Smears were observed in an UTC-8 cell sample shown in lane 3 of FIG.3A and in a cervical carcinoma cell sample shown in lane 4 of FIG. 3A,as in the results when the full sequence was examined in theabove-mentioned (a).

(ii) In the samples of lanes 2 and 3, lanes 4 and 5 of FIG. 3B, smearswere not observed even if the samples were treated with the peptide ofthe present invention, and normal patterns were observed.

(iii) In the samples of lanes 5 and 6, lanes 7 and 8 of FIG. 3A andlanes 6 and 7, lanes 8 and 9 of FIG. 3B, gene expression patterns weresignificantly changed before and after the treatment with the peptide ofthe present invention.

As shown above, it was clarified that a tendency of canceration innormal cells can be determined by comparing changes in gene patternsinduced by treating the cells with the peptide of the present invention.

In particular, when the PCR was performed by using the 11-amino acidprimer (full sequence), a difference in the band patterns was observedin the peripheral blood sample (H.S.) and the skin fibroblast sample,and a difference in a position having a high concentration was observedin the kidney mesangium sample and the pancreatic epithelial sample,though it was a smear. When the PCR was performed by using the 7-aminoacid primer (partial sequence), the band pattern of the pancreaticepithelial sample treated with the peptide for 7 hr was similar to thatof the UTC-8 cell sample used as a control, and a difference in the bandpatterns was observed in the pancreatic epithelial sample by thetreatment with the peptide for 7 hr.

A possible explanation for causes of such significant effects is thatthe UTC-8 cell is a cancer cell of epithelium and the peptide of thepresent invention is derived from UTC-8 cell line.

Therefore, epithelial cells are the most suitable samples for themeasurement of a tendency of canceration using the peptide of thepresent invention, but it was proved that non-epithelial cell samplesincluding peripheral blood, which can be most easily obtained inclinical practice, were useful for the diagnosis of a gene change in thefull sequence.

Example 6

Exhaustive Gene Analysis of 55000 Genes by Using a DNA Chip System(Codelink System)

Characteristics of the peptide of the present invention were clarifiedby conducting the analysis described below in order to examine effectsof the peptide on gene expression of normal cells.

(1) Preparation of Target cRNA

Normal lymphocytes were treated with the peptide of the presentinvention as in Examples 4 and 5, and the gene expression was comparedto that of untreated lymphocytes by using a DNA chip system (CodelinkSystem). In this examination, cRNA derived from bacteria was used as apositive control. Total RNA was extracted by using the QIAGEN RNAextraction kit used in Example 5. The total RNA (5 to 10 μg) wassubjected to reverse transcription using an oligo-dT primer having a T7RNA polymerase promoter region as a reverse primer to generate a pool ofcDNAs complementary to the mRNAs. The condition for the reversetranscription was the same as that in Example 5. After second-strandcDNA synthesis, the treatment with T7 RNA polymerase and biotinylatedrNTPs was performed in vitro transcription (IVT) reaction at 37° C. for14 hr to obtain biotin-labeled cRNA. The amount of the cRNA was measuredwith a spectrophotometer to confirm at least 10 μg of the cRNA.

(2) Hybridization to Codelink Slide

The cRNA (10 μg) obtained in the above (1) was fragmentized and mixedwith hybridization buffer, and then thermally denatured. This mixturesolution was poured into a Codelink slide on which 55000 genes wereimmobilized, and a hybridization reaction was performed at 37° C. for 18hr using an INNOVA 4080 shaker.

(3) Staining and Detection

The hybridization chamber was removed and the plate was washed at 46° C.for 1 hr. The hybridization was detected with Cy5-streptavidin (1:500dilution, at room temperature for 30 min). The plate was further washedat a room temperature for 5 min 5 to 6 times and then washed with 0.05%Tween 20. The plate was dried by using a centrifuge for titer plates.

(4) The plate was scanned with an Arra WORK scanner with a PMT value of600 at a resolution of 10 μm.

(5) The result was analyzed by exclusive software for the Codelinkanalysis. The software used was 1. Batch Submission 2 and 2. CodelinkExpression 2.

Cancer-inducing expression status of cancer-related genes was observedin this experiment too, as in the results shown in Tables 1, 2, and 3.

Example 7

Examination of Antibody Against the Peptide for Cytotoxic Activity onHuman Cancer Cells

Monoclonal antibody (prepared by using BALB/c mouse) against the peptideof the present invention was examined for cytotoxic activity on varioushuman cancer cell lines shown in FIG. 4: Each cancer cell line wasseeded in a 96-well plate at 1×10⁴ cells/well, and cultured in 5% CO₂for 4 days to confirm that the cancer cells proliferated and adhered tothe bottom surface of the plate so as to cover more than 80% of thesurface area. The culture medium was removed by aspiration, and then 30μl of the monoclonal antibody was added to each well under ice-coolingso that the surfaces of the cancer cells were brought into contact withthe culture medium containing the monoclonal antibody. Then, the cancercells were cultured under ice-cooling for 1 hr. As a control, eachcancer cell line was also cultured in PBS buffer not containing themonoclonal antibody. Cytotoxic activity of the monoclonal antibody wasexamined by comparing the cancer cells treated with the monoclonalantibody with those of the control, and viability of the cell wasdetermined by Trypan Blue staining. In the control, no substantialdetachment of the cells from the bottom surface of the plate wasobserved; hence, cytotoxicity was not observed. On the contrary, in thecancer cells treated with the monoclonal antibody against the peptide ofthe present invention, detachment of the cells was observed in cancercells shown in FIG. 4; hence, cytotoxic activity of the monoclonalantibody was observed. In addition, the detachment of the cancer cellswas immediately induced by the addition of the monoclonal antibody, andthe cancer cells were killed by the activity of the monoclonal antibodywithin only one hour. The photograph in the right of FIG. 4 shows humanpancreatic cancer cells stained with FITC-labeled monoclonal antibodyNo. 1 of the present invention. These human pancreatic cancer cells diedafter this treatment.

Example 8

Immunosuppressive Agent Reducing Rejection in Transplantation

The experiment using the DNA chip proved that the peptide (SEQ ID NO: 1)had an ability to activate a cancer-related gene, and further suggestedclinical usefulness of the cellular immunity-inhibition activity of thepeptide. Then, the cellular immunity-inhibition activity of the peptidewas examined from the viewpoint of activity for preventing rejection inskin transplantation.

The peptide of the present invention was administered to mice who havereceived skin transplantation according to the schedule shown in Table6. TABLE 6 Days from Administration Transplantation Transplantation ofPeptide −3 Administration −2 Administration −1 Administration 0Operation 1 2 Administration 3 Administration 4 5 6 7 Administration 8 910 11 12 13 14 Administration 15 16Subject: skin of the back of a C57Black/6N mouse was transplanted to theback of a BALB/c mouseAdministration route: subcutaneously administered into the neckAdministration amount: 50 μg peptide/0.1 ml/mouse Number of the subject:6

The results show that, in the cross-transplantation of skin of the backof a C57Black/6N mouse to the back of a BALB/c mouse, the rejection inthe skin transplantation was suppressed and skin graft survival wasprolonged by intermittently administering the peptide at a very lowamount of 50 μg peptide/0.1 ml/mouse through a subcutaneous route whichis a most gentle administration (see the progress shown by the graph inFIG. 5). Namely, in the control, skin rejection occurred 7 days afterthe skin transplantation and the transplanted skin was rejected and waslost within 10 days after the transplantation in all 6 mice as shown ina photograph (control G1) of mice at bottom right in FIG. 5. On thecontrary, in the mice administered with the peptide, skin rejectionoccurred 12 days after the transplantation; which was a 5 day delay.Furthermore, the transplanted skin of one mouse was survived for 14 daysafter the transplantation as shown in a photograph (treatment G1) atupper right in FIG. 5. Though the transplanted skin was lost in all miceon the 15th day from the date of the transplantation, a delay in therejection in skin transplantation was observed.

Therefore, it was proved that the peptide can be used as animmunosuppressive agent by investigating administration dosage and routeto increase efficiency thereof.

1. A peptide having an ability to activate a cancer-related gene,wherein the peptide is derived from cell membrane surfaces of humansquamous-cell carcinoma cells and comprises the amino acid sequencerepresented by SEQ ID No: 1 or an amino acid sequence includingdeletion, substitution, or addition of one or several amino acids in theamino acid sequence represented by SEQ ID NO:
 1. 2. A peptide having anability to activate a cancer-related gene, wherein the peptide isderived from cell line UTC-8 (FERM BP-08611) established from humansquamous-cell carcinoma; and the peptide shows a detectable peak at adetection wavelength of 214 nm in gel filtration of an extract of thecell line.
 3. An immunosuppressive agent comprising the peptideaccording to claim 1 as an effective component.
 4. An antibody againstthe peptide according to claim
 1. 5. A diagnostic reagent comprising theantibody according to claim 4 for determining tendency of canceration ormalignancy of cancer.
 6. An anticancer agent comprising the antibodyaccording to claim 4 as an effective component.
 7. A polynucleotidecomprising a nucleotide sequence encoding at least three contiguousamino acids of the amino acid sequence of the peptide according toclaim
 1. 8. A diagnostic kit for determining tendency of canceration ormalignancy of cancer, wherein the kit comprises a peptide having anability to activate a cancer-related gene, wherein the peptide isderived from cell membrane surfaces of human squamous-cell carcinomacells and comprises the amino acid sequence represented by SEQ ID No: 1or an amino acid sequence including deletion, substitution, or additionof one or several amino acids in the amino acid sequence represented bySEQ ID NO: 1 and a polynucleotide according to claim
 7. 9. Thediagnostic kit according to claim 8 further comprising at least onemarker gene selected from the group consisting of: Ras oncogene family;v-crk avian sarcoma virus CT10 oncogene homolog-like lactatedehydrogenase B; Placental growth factor; Interleukin 8; MAS1, activatorof S phase kinase; v-raf; v-fms; v-rel; v-src; GRO1; Hepatoma-derivedgrowth factor; Vascular endothelial growth factor; Bone morphogenicprotein 3; Squamous-cell carcinoma antigen recognized by T cell;Interleukin-1 beta; Conserved gene amplified in osteosarcoma; andLymphoid blast crisis oncogene.
 10. A method for manufacturing a peptideaccording to claim 1, the method comprising the steps of: immersingcells prepared by culturing human squamous-cell carcinoma cells in aculture medium into a citrate-phosphate buffer having a pH of 3.3 to 3.4for extraction; and fractionating the extract by gel filtration using acitrate-phosphate buffer having a pH of 6.8 to 7.0 for yielding apeptide having an ability to activate a cancer-related gene in humannormal cells.
 11. An immunosuppressive agent comprising the peptideaccording to claim 2 as an effective component.
 12. An antibody againstthe peptide according to claim
 2. 13. A diagnostic reagent comprisingthe antibody according to claim 12 for determining tendency ofcanceration or malignancy of cancer.
 14. An anticancer agent comprisingthe antibody according to claim 12 as an effective component.
 15. Adiagnostic kit for determining tendency of canceration or malignancy ofcancer, wherein the kit comprises a peptide having an ability toactivate a cancer-related gene, wherein the peptide is derived from cellline UTC-8 (FERM BP-08611) established from human squamous-cellcarcinoma; and the peptide shows a detectable peak at a detectionwavelength of 214 nm in gel filtration of an extract of the cell lineand a polynucleotide according to claim
 7. 16. The diagnostic kitaccording to claim 15 further comprising at least one marker geneselected from the group consisting of: Ras oncogene family; v-crk aviansarcoma virus CT10 oncogene homolog-like lactate dehydrogenase B;Placental growth factor; Interleukin 8; MAS1, activator of S phasekinase; v-raf; v-fms; v-rel; v-src; GRO1; Hepatoma-derived growthfactor; Vascular endothelial growth factor; Bone morphogenic protein 3;Squamous-cell carcinoma antigen recognized by T cell; Interleukin-1beta; Conserved gene amplified in osteosarcoma; and Lymphoid blastcrisis oncogene.
 17. A method for manufacturing a peptide according toclaim 2, the method comprising the steps of: immersing cells prepared byculturing human squamous-cell carcinoma cells in a culture medium into acitrate-phosphate buffer having a pH of 3.3 to 3.4 for extraction; andfractionating the extract by gel filtration using a citrate-phosphatebuffer having a pH of 6.8 to 7.0 for yielding a peptide having anability to activate a cancer-related gene in human normal cells.